The goal of this project is to define the molecular mechanisms involved in the replication of mammalian retroviruses and in particular, to understand the factors which influence the regulated expression of viral genetic information. Studies are being carried out with the murine leukemia virus system. Current interest is focused on the functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT). Mutant and wild-type RT proteins were expressed in E. coli and then purified. The expressed proteins include (i) full-length RT; (ii) an RT (deltaSX) with a deletion of 127 residues immediately upstream of the RNase H domain; (iii) an RT (delta H) missing the entire RNase H domain; and (iv) a chimeric protein having the viral polymerase domain fused to E. coli RNase H. All of the proteins, with the exception of delta H, have RNase H activity. The full-length, chimeric, and delta H proteins have high levels of polymerase activity, whereas the polymerase activity of the deltaSX protein is reduced 200-fold. Interestingly, the deltaSX RNase H activity is also reduced and the characteristic preference for Mn2+ over Mg2+ is lost. This result shows that despite the existence of two separate domains in RT, a mutation in one domain can influence the other. Efforts are now underway to further characterize the RNase H activity of these proteins using substrates which resemble viral intermediates formed during reverse transcription. In other work, translational control of viral gene expression is being investigated by studying readthrough suppression of the UAG codon at the MuLV gag-pol junction. When this UAG is mutated to UAA or UGA, both of these termination codons are suppressed in vitro and in vivo with the same efficiency as UAG. The amino acids inserted in vitro in response to UAA and UGA have been identified: UAA, like UAG, directs the sole incorporation of glutamine,whereas UGA directs the incorporation of arginine, cysteine, and tryptophan. The MuLV suppression system is highly specific and other known UAG and UGA suppressor tRNAs (leucine and serine, respectively) although present in our system, are not functional. This suggests that the "codon context" at the gag-pol junction not only affects the efficiency of suppression, but also determines which tRNAs will be used for suppression. Experiments to elucidate the identity and role of context are in progress.